Cellular responses to Hedgehog (Hh) morphogens culminate in Gli transcription factor activation and the execution of genetic programs associated with tissue patterning, homeostasis and transformation. For example, Hh signaling contributes to formation of the brain, spinal cord, musculature, and skeleton, and pathway dysregulation has been linked to basal cell carcinoma, medulloblastoma, small cell lung cancer, and chronic myelogenous leukemia. Deciphering the molecular mechanisms that control Gli activity state is therefore integral to our understanding of ontogeny and oncogenesis. Several canonical Hh signaling proteins have been identified through mutagenesis or RNA interference screens, including the transmembrane proteins Patched1 (Ptch1) and Smoothened (Smo) and the Gli-interacting protein Suppressor of Fused (Sufu). However, the biochemical and cellular events associated with Gli activation remain enigmatic, particularly those that map downstream of Smo. To gain new insights into this process, we have completed the first genome-scale cDNA overexpression screen for Hh pathway activators, surveying 15,483 mammalian open reading frames (ORFs). Through this gain-of-function screen, cell biological studies, and biochemical analyses, we have discovered that Hipk4, an atypical member of the homeodomain-interacting protein kinase family, acts downstream of Smo to modulate Gli function. Our preliminary studies demonstrate that Hipk4 regulates Gli activity through at least two distinct mechanisms. First, Hipk4 acts in a Smo-independent manner to abrogate the proteolytic processing of Gli factors into transcriptional repressors, generating an intracellular pool of full-length protein that is primed for Hh ligand-dependent activation. Accordingly, Hipk4-overexpressing cells are ultrasensitive to Hh stimulation, and silencing of endogenous Hipk4 by RNA interference inhibits Hh signal transduction. Second, Hipk4 can potentiate cellular responses to exogenous Gli1 or Gli2, as well as elevate the constitutive Hh pathway activity in Sufu null cells, indicating that tis serine/threonine kinase can upregulate the transcriptional activity of full-length Gli proteins to maximize Hh target gene expression. Our findings open a new window into the mechanisms that control Gli function, and discovering the substrates of Hipk4 will reveal some of the key molecular steps in this process. We are now pursuing this goal by integrating a chemical genetic strategy for tagging Hipk4 substrates, mass spectrometry-based sequencing, and various cell biological measures of Hh pathway state. These investigations will advance our basic understanding of Hh signal transduction and foster new strategies for the treatment of Gli-dependent diseases.